Human cardiac troponin I (h.c. TnI) was recently suggested to be a specific and sensitive marker of myocardial cell death. It is released into the blood stream after myocardial damage, e.g. after acute myocardial infarction (AMI) (see e.g. Adams, J. E. et al., Circulation 1993; 88:101-106). Human skeletal troponin I (h.sk.TnI) has been suggested as a sensitive and specific marker of skeletal cell death.
TnI is the subunit of a troponin complex that plays an important role in the Ca2+-dependent regulation of vertebrate skeletal and cardiac muscle contraction. The troponin complex is located on the thin filament of the contractile apparatus and through its association with two other thin filament proteins, actin and tropomyosin, inhibits the actomyosin interaction at submicromolar Ca2+-concentrations, and stimulates interaction at micromolar and higher Ca2+-concentrations. The troponin complex contains three subunits—troponin C (TnC), which is the Ca2+-binding subunit; troponin I (TnI), which is the inhibitory subunit, and troponin T (TnT), which is the tropomyosin binding subunit. The interaction between the three subunits of the troponin complex is so strong that originally the purified troponin was thought to be a single protein.
The Ca2+-dependent regulation is initiated by conformational changes in TnC and subsequent changes in the interaction of TnC with TnI. TnC is composed of two globular domains connected by a central helix. Each domain contains two metal-binding sites. Two sites in the N-terminal domain (sites I and II) bind Ca2+ with low affinity, while sites in the C-terminal domain (sites III and IV) bind Ca2+ with high affinity. At submicromolar concentrations of Ca2+, the metal binding sites in the C-terminal domain of TnC are occupied with Mg2+, whereas the Ca2+-specific sites in the N-terminal domain are empty. In this case the N-terminal region of TnI is bound to the C-terminal domain of TnC, whereas the inhibitory C-terminal regions of the TnI molecule interact with actin and tropomyosin but not with TnC. When the Ca2+-concentration increases up to micromolar level, Ca2+ binds to the N-terminal, Ca2+-specific low affinity sites of TnC. The Ca2+-binding increases the affinity of this domain for the inhibitory and C-terminal regions of TnI, resulting in the release of those fragments from actin and tropomyosin and making strong contact with the extended central and N-terminal regions of TnC. This high-affinity binding of the components of the troponin complex results in important conformational changes of both molecules.
In human striated muscle, three forms of TnI were found: two for skeletal muscle (h.sk. TnI) and one for cardiac muscle (h.c. TnI). The three troponin forms have similar structures, but for human cardiac troponin I the existence of 33 extra amino acids in the N-terminal part of the molecule, as compared to the skeletal TnI isoform, was shown. This extra polypeptide and also some changes in the amino acids make it possible to differentiate human cardiac troponin I from the skeletal forms, for example by immunological methods. All existing diagnostic systems are based on immunological measurement of TnI in serum samples.
It has been shown that in the presence of micromolar and higher concentrations of Ca2+, a mixture of troponins I and C exists in the form of a complex with strong interaction between both molecules. However, when the concentration of Ca2+ in the solution decreases to submicromolar levels, Ca2+ is washed away from the metal binding centers of TnC. After the Ca2+ is removed, the interaction between the two proteins weakens and the conformation of TnI shows resemblance with the native (not complexed) protein.
The concentration of Ca2+ in human serum is high enough for the two low-affinity centers of TnC to bind Ca2+. It means that in the serum of patients with AMI or in patients with various skeletal muscle diseases, a major part of the TnI should be present in the form of a complex with TnC. Our experiments confirmed this hypothesis. We have shown in Katrukha et al. Clinical Chemistry 43:8 1379-1385 (1997), which is included herein as reference, that in the serum of AMI patients, the main part of TnI (50-90%) is presented in the form of a complex with TnC (and probably also with TnT). As mentioned above, the conformation of TnI in complex with TnC at high, i.e. micromolar or higher concentrations of Ca2+ differs from the conformation of free TnI, or of TnI in complex with TnC at submicromolar concentrations of Ca2+.
In common practice highly purified protein is used for the immunization of animals for the production of mono- or polyclonal antibodies. The conformation of purified TnI used for immunization and standard preparation is, however, different from that of TnI in complex with TnC. The change in conformation of the protein can decrease the affinity of the antibodies or make protein-antibody interaction impossible. In addition, due to the strong interaction between the two troponin molecules at micromolar and higher concentrations of Ca2+, TnC will cover part of the surface of the TnI molecule and block some of the epitopes for some antibodies generated by the immunization of animals with highly purified troponin I. As a result, immunoassays based on the use of such antibodies, such as the main part of the commercially available antibodies developed for the said purpose, measure only the free TnI and a part of the TnI in complex with TnC in case the affinity constant of the antibodies is changed due to TnI-TnC interaction; or they measure only the free TnI is case the epitopes of the antibody are completely covered by TnC. In any case the concentration of TnI assayed by such systems in AMI serum samples will be different (lower) than the real concentration of this protein.
Thus there is a need for a method which would improve assays using such antibodies. Such a method would be useful not only in cases of diagnostic systems designed for assaying the cardiac form of TnI in serum but also in immunoassays developed for measurement of skeletal forms of TnI for diagnosis of skeletal muscle tissue necrosis.
In common practice, purified antigen is usually used for the preparation of calibrators or standard preparations for the immunoassays. In case of TnI, the purification is usually a complicated process that takes several days and includes several steps of column chromatography (affinity, ion exchange, etc.). It is a well known fact that TnI is highly susceptible to proteolysis. During long-term purification, the troponin I molecule can be partially cleaved by proteases and thus some part of the epitopes for some antibodies can be lost. In addition, all existing methods for TnI purification include stages in which highly concentrated (6-8M) urea solutions are used to dissociate the components of the troponin complex. Such rigid treatment can lead to irreversible changes in the conformation of some parts of the TnI molecule. Consequently the conformation of the TnI molecules in highly purified preparations can be different from that of the native protein. In addition, long-term contact of the protein with urea at high concentrations can result in a partial carbamylation of the TnI molecule. Thus the immunological activity of highly purified TnI can be different from that of native TnI.